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Abstract:

The present invention provides for the use of iota- and/or
kappa-carrageenan for the manufacture of an antiviral pharmaceutical
composition for the prophylaxis or treatment of a pathological condition
or disease caused by or associated with an infection by a respiratory
virus selected from the group consisting of orthomyxovirus,
paramyxovirus, adenovirus and coronavirus, The present invention further
provides for the use of fucoidan, in particular of high molecular weight
fucoidan, for the manufacture of an antiviral pharmaceutical composition
for the prophylaxis or treatment of a pathological condition or disease
caused by or associated with an infection by a respiratory virus selected
from the group consisting of orthomyxovirus and paramyxovirus.

Claims:

1. A method of prophylactic or therapeutic treatment of a symptom,
condition or disease caused by or associated with an infection by a
respiratory virus selected from the group consisting of paramyxovirus,
human influenza A virus, and adenovirus of subtype B, comprising
administering to a subject a pharmaceutical composition comprising
iota-carrageenan as an antiviral active ingredient in an antiviral
effective amount.

2. The method according to claim 1, wherein the respiratory virus is a
virus that enters a host cell via receptor-mediated attachment to the
cell, the receptor comprising one or more sugar residues.

3. The method according to claim 1, wherein said respiratory virus is a
paramyxovirus.

4. The method according to claim 1, wherein said respiratory virus is a
human influenza A virus.

5. The method according to claim 4, wherein said virus binds to the host
cell via a cell surface receptor that comprises sialic acid residues
having alpha 2-6 linkages.

6. The method according to claim 1, wherein said respiratory virus is an
adenovirus of subtype B.

8. The method according to claim 7, wherein said paramyxovirus is RSV.

9. The method according to claim 1, wherein said pathological condition
or disease is selected from the group consisting of acute bronchitis,
chronic bronchitis, rhinitis, sinusitis, croup, acute bronchiolitis,
pharyngitis, tonsillitis, laryngitis, tracheitis, asthma and pneumonia.

10. The method according to claim 1, wherein said symptom is selected
from the group consisting of fever, pain, dizziness, shivering, sweating,
and dehydration.

11. The method according to claim 1, wherein the antiviral pharmaceutical
composition is administered topically on skin or mucosa in the form of
one of a skin lotion, cream, ointment, gel and powder, including a powder
for inhalation, spray, foam, liquid drops, or a gargle solution.

12. The method according to claim 1, wherein the antiviral pharmaceutical
composition is administered orally in the form of a liquid solution, or
in the form of a semi-solid or solid preparation such as a dry powder,
tablets, capsules, or dragees.

13. The method according to claim 11, wherein the composition is liquid
or semi-solid and comprises as a ready-for-use preparation
iota-carrageenan in an amount of between 0.01% and 10% by weight,
relative to the total volume of the preparation.

14. The method according to claim 12, wherein the composition is solid
and comprises as a ready-for-use preparation iota-carrageenan in an
amount of between 0.01% and 10% by weight.

15. The method according to claim 1, wherein the composition further
comprises kappa-carrageenan.

16. The method according to claim 15, wherein the composition comprises
50% or more by dry weight of iota-carrageenan, relative to the total dry
weight of carrageenans present in the composition.

17. The method according to claim 1, wherein said composition further
comprises at least one pharmaceutically acceptable carrier and/or
additive.

19. The method according to claim 18, wherein said pharmaceutically
acceptable salt is present in the composition in an amount of 1% or less.

20. The method according to claim 1, wherein the composition comprises a
part or all of iota-carrageenan by way of its salt.

21. The method according to claim 1, wherein the composition is sterile.

22. The method according to claim 1, wherein prior to the administering
the composition is coated or impregnated onto a solid surface of a
hygiene or sanitary item and the administering comprises the subject
contacting the coated or impregnated surface of the hygiene or sanitary
item.

23. The method according to claim 1, wherein prior to the administering
the composition is integrated in a lipstick and the administering
comprises the subject contacting the lipstick.

24. The method according to claim 1, wherein the composition further
comprise a non-carrageenan physiologically active compound, said compound
as the main active ingredient.

25. The method according to claim 24, wherein the composition comprises
iota-carrageenan as an anti-paramyxoviral or anti-orthomyxoviral adjuvant
together with one or more non-carrageenan physiologically active
substances that are applied to the prophylaxis or therapy of infectious
and/or inflammatory diseases, allergies and/or conditions of an impaired
or suppressed immune system.

26. The method according to claim 1, wherein the subject is an individual
being a high-risk patient selected from the group consisting of a
COPD-patient, an asthma patient, a person with allergies, a person with
impaired immune, cardiac, or pulmonary system, and a transplantation
patient.

27. The method according to claim 12, wherein the composition is liquid
or semi-solid and comprises as a ready-for-use preparation
iota-carrageenan in an amount of between 0.01% and 10% by weight,
relative to the total volume of the preparation.

28. The method according to claim 22, wherein the sanitary item is a
hygiene or sanitary glove, tissue or paper, a nasal tissue or paper, a
cotton swab, dust mask or sanitary or medical facial mask.

Description:

[0001] This is a Continuation of application Ser. No. 12/673,145 filed
Feb. 24, 2010, which in turn is a Continuation of Application No.
PCT/EP2008/006910 filed Aug. 22, 2008, which claims the benefit of U.S.
Provisional Application No. 60/935,668 filed Aug. 24, 2007. The
disclosures of the prior applications are hereby incorporated by
reference herein in their entirety.

TECHNICAL FIELD

[0002] The present invention relates to sulfated polysaccharides selected
from the group of carrageenans and fucoidans and pharmaceutical
compositions made thereof, wherein said sulfated polysaccharides are
present as antiviral active ingredients, for medical or veterinary use in
the prevention or treatment of diseases caused by or associated with a
virus entering an individual's body via the respiratory tract, the virus
being selected from the group of orthomyxoviridae, paramyxoviridae,
adenoviridae and coronaviridae.

BACKGROUND OF THE INVENTION

[0003] Orthomyxoviridae are RNA viruses, the most prominent members being
influenza virus species. Influenza A and B virus are the two types of
influenza viruses that cause epidemic human disease. They are typically
spread from person to person, primarily through respiratory droplet
transmission.

[0004] The paramyxoviridae family includes respiratory syncytial virus
(RSV), parainfluenza virus and metapneumavirus. RSV and parainfluenza
virus infections may cause serious respiratory infections in infants and
young children but may also cause severe disease in elderly persons and
adults having an impaired immune system.

[0005] Among the human parainfluenza virus species four members are known
to cause serious respiratory diseases in children for which presently no
effective prevention or therapy is available, the four members being
parainfluenza virus types 1 through 4. Parainfluenza virus and RSV
outbreaks are believed to contribute substantially to increased
hospitalization and mortality rates. Patients with impaired immune,
cardiac, or pulmonary systems are at increased risk of running into
serious complications following a paramyxovirus infection and will
therefore particularly benefit from an antiviral therapy.

[0006] Respiratory syncytial virus (RSV), which is the most wide-spread
respiratory pathogen typically afflicting individuals during infancy and
early childhood worldwide, causes yearly outbreaks of pneumonia and
bronchiolitis during winter time and early in spring. RSV bronchiolitis
and pneumonia require hospitalization of hundreds of thousands of infants
every year. Passive protection against RSV is available at least in part
at a reported 55% success rate upon monthly intramuscular injection of
the humanized monoclonal anti-RSV antibody palivizumab (Synagis®).

[0007] The coronaviridae family includes coronavirus and torovirus.
Coronaviruses are known to infect the upper respiratory tract and the
gastrointestinal tract in mammals and birds. It is believed that
coronaviruses cause a high percentage of all common colds in human
adults.

[0008] Adenoviridae are DNA viruses which typically infect the upper
respiratory tract. At present, more than 50 different serotypes of human
adenovirus are known, grouped in six subtypes A to F, which are
responsible for 5-10% of upper respiratory infection in children.

[0009] In view of the aforementioned it is believed that there exists a
strong need for a potent antiviral pharmaceutical composition that is
easily applicable and effective in the prevention or treatment of
respiratory viral infections caused by members of the paramyxoviridae,
orthomyxoviridae, coronaviridae and/or adenoviridae families.

[0010] Sulphated polysaccharides including carrageenans and fucoidan have
been known for their antiviral efficacy for decades. Accordingly, the
prior art is replete with scientific articles on the antiviral effects of
carrageenans. In a most interesting review, Gonzalez M. E. et al. (1987,
Antimicrob. Agents Chemother. 31, 1388-1393) report an antiviral efficacy
of different sulphated polysaccharides including iota-carrageenan against
several animal viruses. Iota-carrageenan showed antiviral activity
against the enveloped viruses HSV-1, HSV-2, Semliki Forest virus (SFV),
vaccinia virus and African swine fever virus (ASF) and against the naked
encephalomyocarditis (EMC) virus. Iota-carrageenan had no effect on the
enveloped viruses vesicular stomatitis virus (VSV) and measles virus and
on the naked viruses polio virus type 1 and adenovirus type 5.

[0012] WO 88/06396 discloses a method for the treatment of retroviral
infections, including infection with HIV, by administering a carrageenan
or a mixture of carrageenans.

[0013] Girond et al. (1991, Research Virol. 142, 261-270) disclose that
sulphated polysaccharides like iota-, kappa-, and lambda-carrageenan have
an effective inhibitory activity against the replication of hepatitis A
virus (HAV).

[0015] EP 0293826 discloses the therapeutic and prophylactic application
of sulfated polysaccharides such as fucoidan and carrageenans to inhibit
HIV-1 in vitro.

[0016] U.S. Pat. No. 5,658,893 discloses a method for inhibiting rotavirus
infection of human cells by contacting the rotavirus with
lambda-carrageenan. It is further disclosed that iota- and
kappa-carrageenan did not exhibit anti-rotaviral activity.

[0017] US 2003/181415 A discloses that sulphated polysaccharides,
including cellulose sulphate, are known to be effective against various
enveloped viruses and in particular against herpes simplex virus (HSV),
Papilloma viruses and HIV.

[0018] Fucoidan is a sulphated polysaccharide mainly extracted from
various species of brown seaweed. There are two types of pharmacological
grade fucoidans available on the market, a high molecular weight fucoidan
(HMWF) fraction having an average molecular weight ranging from about
1,000,000 to 2,000,000 Da (e.g. Kraeber, Germany) and a low molecular
weight fucoidan (LMWF) fraction having an average molecular weight of
8,200 Da.

[0019] F-fucoidan is mainly composed of sulphated esters of fucose, while
U-fucoidan is comprised of about 20% of glucuronic acid.

[0020] Carrageenan is a generic term for linear sulphated galactose-based
polysaccharides extracted from seaweed (rhodophyceae). It is mainly used
as a thickener, gelling agent, stabilizer or emulsifier in pharmaceutical
and food products. There exist more than 10 structurally different
carrageenans, their nature depending on the seaweed genus from which they
are extracted. The three main types are iota-, kappa- and
lambda-carrageenan, which differ slightly in their structure and degree
of sulphatation. Iota-carrageenan is a soft-gel forming sulphated
galactan predominantly extracted from red seaweed Gigartina stellata and
Chondrus crispus. Kappa-carrageenan yields strong, rigid gels and is
predominantly produced from Kappaphycus cottonii. Lambda-carrageenan,
which is the most common form, is frequently used to thicken dairy
products.

[0021] Despite the long known antiviral activity of carrageenans against
viruses such as, e.g. HIV, HSV, HAV, HTLV, or HPV, the mechanism of how
carrageenans exhibit antiviral activity still needs further
clarification.

[0022] For example, Baba M. et al. (1988, Antimicrob. Agents Chemother.
32, 1742-1745) speculate that sulphated polysaccharides including kappa-
and lambda-carrageenan inhibit or at least contribute to the inhibition
of virus adsorption of several enveloped viruses to the host cell
surface. Similarly, US 2005/0261240 assumes that carrageenan may
non-specifically bind to a virus thereby blocking virus receptor sites.
Damonte E. B. et al. (2004, Curr. Med. Chem. 11, 2399-2419) disclose that
sulphated polysaccharides may mimic cellular heparin sulphate and
therefore block viral receptor sites responsible for the initial
interaction between virus and host cell, whereas Gonzalez M. E. et al.
(1987, Antimicrob. Agents Chemother. 31, 1388-1393) found, using labeled
virion particles, that HSV-1 virions are internalized even in the
presence of high concentrations of iota-carrageenan. They suggest that at
least for HSV-1 carrageenan inhibits a step in virus replication
subsequent to virus attachment and entry into the cell but still prior to
the synthesis of late viral proteins.

[0023] Turner E. V. and Sonnenfeld G. (1979, Infection and Immunity 25,
467-469) disclose an antiviral activity of lambda- but not
kappa-carrageenan against bovine vesicular stomatitis virus which
antiviral activity is due to immunomodulation, i.e. to the induction of
interferon.

[0024] In conclusion, it can be summarized taking the words of U.S. Pat.
No. 5,658,893, that "in view of the different responses by different
viruses to sulphated polysaccharides, it is clear that the response of a
particular virus to carrageenan cannot be predicted with certainty
without experimentation. The mechanism by which sulphated
polysaccharides, particularly the carrageenans, inhibit viral replication
and infectivity may not be uniform as different investigators reported
contradictory findings when working with different viruses and cell
types. It would not be obvious to one skilled in the art that a substance
such as a sulphated polysaccharide that is an effective inhibitor of one
virus would demonstrate similar efficacy against another virus."

[0025] In the light of the above, the present invention now provides for a
carrageenan- and/or fucoidan-based antiviral composition suitable in the
prophylactic or therapeutic treatment of respiratory viral infections
caused by members of the paramyxoviridae, orthomyxoviridae, adenoviridae
and/or coronaviridae families. Contrary to the disclosure of Fujisawa H.
et at. (1987, J. gen. Virol. 68, 425-423), who report that intranasal
administration of carrageenan did not reduce the influenza A virus titer
in mice but on the contrary even enhanced the animals' susceptibility to
the virus depending on the amount of virus used for infection, it was now
surprisingly found that carrageenan, in particular iota- and
kappa-carrageenan, has indeed antiviral efficacy against various members
of the orthomyxoviridae, paramyxoviridae, adenoviridae and coronaviridae.

[0026] Additionally, it was surprisingly found that fucoidan, particularly
the high molecular weight fucoidan (HWMF) fraction, has antiviral
efficacy against various members of the orthomyxoviridae and
paramyxoviridae.

[0027] Accordingly, the present invention aims at providing an antiviral
pharmaceutical composition suitable for the prevention or treatment of
respiratory tract infections caused by a virus selected from the group
consisting of orthomyxovirus, paramyxovirus, adenovirus and coronavirus,
as well as for diseases or pathological conditions associated with such
primary viral infections, such diseases or conditions comprising
secondary viral or bacterial infections as well as bodily symptoms
typically associated with any such primary or secondary infection
including symptoms such as fever, pain, dizziness, shivering, sweating,
and/or dehydration.

DESCRIPTION OF THE INVENTION

[0028] In a first embodiment the present invention relates to the use of
carrageenan as an antiviral active ingredient in the manufacture of a
pharmaceutical composition for the prophylaxis or treatment of a
pathological condition or disease caused by or associated with an
infection by a respiratory virus, wherein said respiratory virus
comprises at least one protein attached to or integrated in its viral
membrane, the protein capable of binding to a host cell via a receptor
having at least one sugar moiety, the respiratory virus being selected
from the group consisting of orthomyxovirus and paramyxovirus and the
carrageenan being selected from the group consisting of iota- and
kappa-carrageenan.

[0029] A "host cell" referred to herein is any eukaryotic cell that is
naturally, i.e. under natural or natural-like conditions, targeted and
penetrated by any of the viruses referred to hereinafter. For
experimental and laboratory purposes it is state of the art to use host
cell lines that are typically recognized as suitable in vitro models for
testing the efficiacy of physiologically active agents and which allow
for at least some predictability of the results with regard to comparable
human applications.

[0030] The present invention further relates to a pharmaceutical
composition comprising iota- and/or kappa-carrageenan as an antiviral
active ingredient and to applications thereof in the prevention or
treatment of diseases caused by or associated with a virus entering an
individual's body via the respiratory tract, wherein said virus comprises
at least one protein attached to or integrated in its viral membrane, the
protein capable of binding to a host cell via a receptor having at least
one sugar moiety, and the virus being selected from the group consisting
of orthomyxovirus and paramyxovirus.

[0031] The term "antiviral active ingredient" as used herein refers to a
compound that is directly or indirectly effective in specifically
interfering with at least one viral action selected from the group
consisting of virus penetration of eukaryotic cells, virus replication in
eukaryotic cells, virus assembly, virus release from infected eukaryotic
cells, or that is effective in unspecifically inhibiting a virus titer
increase or in unspecifically reducing a virus titer level in a
eukaryotic or mammalian host system. It also refers to a compound that
prevents from or reduces the likelihood of getting a viral infection.

[0032] The present pharmaceutical composition may be administered, as the
case may be, before or after the onset of a viral infection, i.e. for
prophylactic or therapeutic treatment purposes, or for both prophylactic
and therapeutic administration.

[0033] The term "prophylaxis" or "prophylactic treatment" as used herein
relates to the administration of the present pharmaceutical composition
to a healthy individual in order to reduce said individual's
susceptibility for a viral infection.

[0034] The term "therapy" or "therapeutic treatment" as used herein
relates to the administration of the present pharmaceutical composition
in order to achieve a reduction in severity and/or frequency of symptoms,
elimination of symptoms and/or underlying cause, prevention of the
occurrence of symptoms and/or their underlying cause, and/or improvement
or remediation of damage directly caused by or indirectly associated,
e.g. through secondary infection, with the viral infection.

[0035] It is known that orthomyxo- and paramyxoviruses and several other
viruses attach to the host cell via binding to receptors present on the
host cell surface, the receptors typically being glycoproteins or
glycolipids containing sugar residues including sialic acid (N-actyl
neuramic acid) and heparan sulphate residues.

[0037] The cellular uptake of a paramyxovirus involves at least two of its
integral membrane glycoproteins. One of them, typically selected from the
group of glycoproteins HN, H and G, is involved in cell attachment and
the other glycoprotein, i.e. glycoprotein F, is involved in mediating
pH-independent fusion of the viral envelope with the membrane of the host
cell. For example, respiroviruses and rubulaviruses bind to sialic acid
residues of glycoprotein or glycolipid host cell receptors.

[0038] The attachment of RSV is not fully understood but attachment to the
host cell most likely involves interaction with heparan sulphate, a
glucoseaminoglycan that is part of the extracellular matrix.

[0039] Along with revealing the present invention it was observed in vitro
that exposing host cells to carrageenan, in particular iota- and/or
kappa-carrageenan, may alter the structure of the cell surface of a said
host cell, presumably by causing conformational changes in the three
dimensional structure of certain cell surface receptors or by masking
and/or modifying said receptors.

[0040] Surprisingly, this change on the host cell surface prevented
orthomyxo- and paramyxoviruses from attachment to the host cells even
after removal of the carrageenan from the host cells, i.e. in the absence
of carrageenan at the time of artificially triggering an infection by
inoculation in vitro.

[0041] This particular antiviral effect of carrageenan was observed as
long as the artificially triggered infection was initiated no later than
about two hours after removal of the polymer from the host cells,
indicating a relatively long lasting effect of the structural changes on
the cell surface caused by interaction with carrageenan.

[0042] In contrast to paramyxo- and orthomyxoviruses, a protective effect
in the absence of carrageenan at the time of triggering infection was not
observed for other viruses including rhinovirus and coronavirus, while
protection was only achieved in the presence of carrageenan.

[0043] Therefore and in view of the experimentally proven antiviral
efficacy of carrageenan against rhinovirus infection, it is concluded
that with rhinovirus and probably other viruses, too, the antiviral
effect may be due to attachment of the carrageenan polymer to the virions
rather than to chemical or physical interaction of the polymer with the
host cell receptors.

[0044] As mentioned herein before the carrageenan compositions of the
present invention may successfully be applied against orthomyxovirus
infection, including infections caused by influenza A or B virus, and
especially wherein said influenza A or B virus is a human virus that
binds to a host cell via 2-6 sialic acid linkage. Human influenza virus
infections are best treated using predominantly or solely
iota-carrageenan or fucoidan as the antiviral active ingredients.

[0045] Carrageenan may, however, also be successfully applied against
infections wherein said influenza A virus is an avian virus that binds to
a host cell via 2-3 sialic acid linkage. In this latter case,
kappa-carrageenan turned out to be the most efficacious carrageenan, the
use of which is therefore preferred in connection with avian virus
prophylaxis or therapeutic treatment. Whereas iota-carrageenan and
fucoidan were found to be much less active or even inactive towards avian
influenza A virus.

[0046] The paramyxovirus species found to be susceptible to carrageenan
treatment are selected from the group consisting of human parainfluenza
virus (HPV) type 1, HPV type 2, HPV type 3, HPV type 4 and RSV. Where the
selected paramyxovirus is RSV it is preferred that iota-carrageenan or
fucoidan be applied as a predominant or sole sulfated polysaccharide
ingredient for prophylactic or therapeutic administration, because
iota-carrageenan and fucoidan yielded the best results in the
experimental set-ups.

[0048] Carrageenan is especially suitable for topical application to treat
skin or mucosal inflammation. Carrageenan useful for topical application
to the skin or mucosa in accordance with the present invention has a
molecular weight ranging from about 15,000 to 5,000,000 Da, fractions
having average molecular weights of more than 50,000 Da, and especially
fractions having average molecular weights in the range of from 50,000 to
3,000,000 Da being particularly preferred.

[0049] Systemic, e.g. parenteral or oral administration is possible, too,
especially using lower molecular weight polymers having an average
molecular weight in the 15,000 to 100,000 Da range.

[0050] The pharmaceutical composition according to the present invention
adjusted for parenteral use may be provided as a preparation selected
from the group of skin lotions, creams, ointments, gels, powders
including powders for inhalation, sprays, foams, liquid drops or gargle
solutions. The carrageenan preparations may, however also be adjusted for
oral administration, e.g. as liquid solutions, or semi-solid or solid
preparations such as dry powders, tablets, capsules, dragees or any other
orally ingestible galenic form. Pharmaceutical compositions for mucosal
application include nose sprays or drops and any other nasal drug
delivery system known in the art such as disclosed, for example, in U.S.
Pat. No. 6,391,452.

[0051] Where the composition is for topical use and is liquid or
semi-solid it typically comprises in its ready-for-use form carrageenan
in an amount of between 0.01 and 10%, preferably between 0.01 and 5%,
most preferably between 0.1 and 2% by weight, relativ to the total volume
of the composition. Where the composition is solid it typically comprises
in its ready-for-use form carrageenan in an amount of between 0.01 and
10%, preferably between 0.01 and 5%, most preferably between 0.1 and 2%
by weight relative to the total weight of the composition.

[0052] The carrageenans useful in the present invention are commercially
available but may also be prepared by extraction from seaweed plants
pursuant to extraction procedures known in the art.

[0053] If not indicated otherwise the term "carrageenan" as used herein
refers to either iota-, or kappa-carrageenan or a mixture of both.

[0054] The carrageenan of the present invention can further be a
homopolymer or a heteropolymer. A carrageenan homopolymer is built of
subunits of only one kind of either iota-, or kappa-carrageenan, whereas
a carrageenan heteropolymer comprises subunits of both said carrageenans.

[0055] A "mixture" of carrageenans may thus also refer to a composition of
matter comprising a mixture of different carrageenan subunits as part of
at least one heteropolymeric carrageenan present in said composition.

[0056] Typically, the antiviral pharmaceutical compositions according to
the present invention are substantially free of carrageenans other than
iota- and kappa-carrageenan, i.e. comprise either iota- or
kappa-carrageenan, or a mixture of both, in an amount of 80% or more,
preferably of 90% or more, and especially of up to 99% (w/w) or more,
relative to the dry weight of all carrageenans present in the
composition.

[0057] For some applications the composition is substantially comprised
solely of iota-carrageen, while for other applications the composition
may comprise more than 50%, preferably more than 70%, and especially up
to more than 95% (w/w) by dry weight of iota-carrageenan, relative to the
total dry weight of all carrageenans present in the composition.

[0058] For still other applications it may be useful that the composition
comprises more than 50%, preferably more than 70%, and especially up to
more than 95% (w/w) by dry weight of kappa-carrageenan, relative to the
total dry weight of all carrageenans present in the composition.

[0059] Where the composition comprises at least one heteropolymeric
carrageenan the numeric values given above relate to the percentages of
the respective carrageenan subunits.

[0060] The antiviral carrageenan compositions in accordance with the
present invention may further comprise at least one pharmaceutically
acceptable carrier and/or additive suitable and permitted for medical
application.

[0061] The carrier may be a diluent, e.g. water, saline, optionally
phosphate buffered saline (PBS), an excipient, or another vehicle which
facilitates the administration of the composition. Where the composition
is solid, semi-solid or fluid the groups of carriers and additives,
respectively, may comprise but are not limited to SiO2, TiO2, a
binder such as microcrystalline cellulose, polyvinylpyrrolidone, gum
tragacanth, gelatine, starch, lactose, lactose monohydrate, alginic acid
or maize starch; a lubricant or surfactant like magnesium stearate or
sodium lauryl sulphate; a glidant like e.g. colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin.

[0063] For many applications it is useful that the composition comprises
sodium chloride as an additive.

[0064] Typically, a pharmaceutically acceptable salt is present in the
composition in an amount of not more than 1%, preferably not more than
0.6% (w/v).

[0065] In the compositions of the present invention the carrageenan itself
may be present as a salt, too, preferably as a sodium salt.

[0066] It is preferred that the present compositions for prophylactic or
therapeutic medical application be provided as sterile, germ-free,
pathogen-free, pyrogen-free and/or allergen-free preparations.

[0067] Carrageenan was found to be non-toxic upon oral or dermal
administration, or upon inhalation, even when applied at extremely high
doses. It was therefore classified as "generally recognized as safe"
(GRAS) by the Food and Drug Administration (FDA).

[0068] Carrageenan, especially iota-carrageenan, can also be used as an
antiviral agent effective against orthomyxovirus and paramyxovirus
infections in various medicinal products together with other
physiologically active compounds or drugs as the main active ingredients,
wherein the role of carrageenan includes its use as a carrier or as an
additive such as an emulsifier or a viscosity modifying agent. It is
compatible with most pharmaceutical preparations without causing
undesired side-effects,

[0069] It is therefore within the scope of the present invention to
provide pharmaceutical compositions comprising carrageenan as an
additive, more specifically as an antiviral, particularly
anti-paramyxoviral or anti-orthomyxoviral, adjuvant to medicaments that
are typically applied in the prophylaxis or therapy of infectious and/or
inflammatory diseases, allergies and/or conditions of an impaired or
suppressed immune system. In particular, it is within the scope of the
present invention to use carrageenan, preferably iota-carrageenan, as an
anti-influenza virus effective adjuvant in combination with medicaments
useful in the prophylaxis or therapy of infectious and/or inflammatory
diseases, allergies and/or conditions of an impaired or suppressed immune
system.

[0070] It was observed that carrageenan may also have immuno-modulating
activity, particularly immune system strengthening activity. It is not
yet clear, however, whether this activity is directly associated with a
physiological interference of the substance or indirectly through its
antiviral efficacy.

[0071] The composition of the present invention will usually be formulated
into preparations for topical or mucosal use, preferably selected from
sprays, particularly nose sprays, powders, drops, gargle solutions,
foams, gels, creams, ointments, lotions, lozenges, and the like. However,
the pharmaceutical composition of the present invention may also be
coated onto solid surfaces of hygiene or sanitary items such as, for
example, facial hygiene or sanitation articles that are typically used in
the oral and/or nasal areas including but not limited to nasal tissues or
papers, and handkerchiefs.

[0072] More specifically, the pharmaceutical composition may be applied,
e.g. sprayed--much like disinfectants--onto gloves, hygiene tissues or
papers including nasal tissues or papers, in order to exert a virucide
effect at least to some extent, thus contributing to reducing an
individual's repeated self-infection by contaminated fingertips and also
to reduce viral distribution among different individuals that are in
close, e.g. hand-to-hand, contact with each other.

[0073] Further items coated, impregnated or soaked with a pharmaceutical
composition on a carrageenan basis comprise cotton swabs, dust masks or
sanitary or medical facial masks. Even lipsticks can be formulated to
contain an antiviral effective amount of carrageenan. These hygiene or
sanitation articles can be used prophylactically or for therapeutical
treatment against a viral infection and may assist in the prevention or
reduction of a risk of infection.

[0074] It is an objective of the present invention to provide for
carrageenan-based antiviral compositions for the prophylaxis or
therapeutic treatment of individuals especially susceptible to or at
increased risk of orthomyxo- or paramyxovirus infection including
high-risk patients selected from the group consisting of COPD-patients,
asthma patients, individuals suffering from allergies, or from impaired
immune, cardiac, or pulmonary systems, and transplantation patients.

[0075] In accordance with the present invention carrageenan may also be
used as an antiviral active ingredient in the manufacture of a
pharmaceutical composition for the prophylaxis or therapeutic treatment
of a pathological condition or disease caused by or associated with an
infection by a respiratory virus being selected from the group consisting
of adenovirus and coronavirus, the adenovirus preferably being adenovirus
type B (Ad50).

[0076] Adenovirus and coronavirus infections are best treated using
compositions where iota-carrageenan is the sole or predominant active
antiviral ingredient.

[0077] Experimental trials further confirmed that also fucoidan is
usefully applied as an antiviral active ingredient in the manufacture of
a pharmaceutical composition effective for the prophylaxis or treatment
of pathological conditions or diseases caused by or associated with a
respiratory virus infection, the respiratory virus selected from the
group consisting of orthomyxo- and paramyxoviruses. In this context, the
orthmyxovirus is typically influenza virus A or B, and the paramyxovirus
is typically RSV.

[0078] A fucoidan-based composition in its ready-for-use form for topical
or oral administration may comprise fucoidan in an amount of between 0.01
and 10%, preferably between 0.01 and 5%, most preferably between 0.1 and
2% weight per volume (w/v) or weight per weight (w/w) depending on
whether the composition is liquid or semi-solid (w/v-percentages) or
solid (w/w-percentages). It may further comprise at least one
pharmaceutically acceptable carrier and/or additive, as well as other
physiologically active substances or drugs. For example, sodium chloride
is frequently used as an additive. In general, pharmaceutically
acceptable salts may be present in the fucoidan compositions in an amount
of not more than 1%, preferably not more than 0.6%.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079]FIG. 1 shows the efficacy of iota-carrageenan in reducing plaque
formation of influenza A/Chile/1/93 H1N1 virus in MDCK cells at different
doses ranging from a final concentration of 0.1 to 100 μg/ml.

[0080] Ordinate=percentage of plaque formation after infection of MDCK
cells with influenza A/Chile/1/93 H1N1 virus suspension containing
different concentrations of iota-carrageenan relative to the plaque
formation of MDCK cells infected with influenza A/Chile/1/93 H1N1 virus
suspension without iota-carrageenan (set as 100%); abscissa=different
final concentrations of iota-carrageenan in the virus suspension in
μg/ml.

[0081]FIG. 2 shows the efficacy of iota-carrageenan in reducing plaque
formation of influenza A/Aichi2/68 H3N2 virus in MDCK cells at different
doses ranging from a final concentration of 75 to 300 μg/ml.

[0082] Ordinate=percentage of plaque formation after infection of MDCK
cells with influenza A/Aichi2/68 H3N2 virus suspension containing
different concentrations of iota-carrageenan relative to the plaque
formation of MDCK cells infected with influenza A/Aichi2/68 H3N2 virus
suspension without iota-carrageenan (set as 100%); abscissa=different
final concentrations of iota-carrageenan in the virus suspension in
μg/ml.

[0083]FIG. 3 shows the efficacy of iota-carrageenan in reducing plaque
formation of parainfluenza virus 3 in Hep-2 cells at different doses
ranging from a final concentration of 0.1 to 100 μg/ml.

[0084] Ordinate=percentage of plaque formation after infection of Hep-2
cells with parainfluenza virus 3 suspension containing different
concentrations of iota-carrageenan relative to the plaque formation of
Hep-2 cells infected with parainfluenza virus 3 suspension without
iota-carrageenan (set as 100%); abscissa=different final concentrations
of iota-carrageenan in the virus suspension in μg/ml.

[0085] FIG. 4 shows the efficacy of iota-carrageenan pretreatment in
reducing plaque formation of parainfluenza virus 3 in HeLa cells at
different doses ranging from a final concentration of 13 to 400 μg/ml.

[0086] Ordinate=percentage of plaque formation after infection of
pretreated HeLa cells (preincubation for 3 h with iota-carrageenan;
subsequent removal of the polymer) with parainfluenza virus 3 suspension
relative to the plaque formation of untreated HeLa cells (without
iota-carrageenan preincubation) (set as 100%); abscissa=different final
concentrations of iota-carrageenan in the preincubation medium in
μg/ml. C=cells preincubated with polymer carboxymethylcellulose
(negative control).

[0088] Ordinate=percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
carrageenan in the virus suspension (FIG. 5A) or in the preincubation
medium (FIG. 5B) in μg/ml; FIG. 5A=cells infected with feline
coronavirus FIP in the presence of iota-, kappa- or lambda-carrageenan;
FIG. 5B=cells preincubated for 3 h with iota-, kappa- or
lambda-carrageenan, subsequently washed three times with PBS and infected
with feline coroanvirus FIP in the absence of the polymer.

[0090] Ordinate=percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-carrageenan in the virus suspension (FIG. 6A) or in the
preincubation medium (FIG. 6B) in μg/ml; FIG. 6A=cells infected with
human rhinovirus type 8 (HRV8) in the presence of iota-carrageenan; FIG.
6B=cells preincubated for 3 h with iota-carrageenan, subsequently washed
three times with PBS and infected with HRV8 in the absence of
iota-carrageenan.

[0091]FIG. 7 shows the binding of FITC-labelled iota-carrageenan to HeLa
cells after successive washing with PBS.

[0092] Ordinate=calculated iota-carrageenan (converted from fluorescent
light units into μg/ml using a standard curve); abscissa=washing steps
of the cells with PBS; 0=FITC-labelled iota-carrageenan containing
supernatant was removed from the cells; 1=first washing step of the cells
with PBS; 2=second washing step of the cells with PBS; 3=third washing
step of the cells with PBS.

[0094] Ordinate=percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-carrageenan in the virus suspension (dark bars) or in the
preincubation medium (light bars) in μg/ml; dark bars=cells infected
with RSV in the presence of iota-carrageenan; light bars=cells
preincubated for 3 h with iota-carrageenan, subsequently washed three
times with PBS and infected with RSV in the absence of iota-carrageenan.

[0095]FIG. 9 shows the results of an RSV induced cell death inhibition
experiment in HNep cells.

[0096] Ordinate=percentage of uninfected cells after infection and 5 days
of incubation relative to uninfected control cells; abscissa=time points
for addition of iota- carrageenan in a final concentration of 100
μg/ml to the incubation medium (hours post infection).

[0098] Ordinate=percentage of uninfected cells after infection and 5 days
of incubation relative to uninfected control cells; abscissa=different
final concentrations of iota- carrageenan in the virus suspension in
μg/ml.

[0100] Ordinate=percentage of uninfected cells after infection and 6 days
of incubation at 37° C. relative to uninfected control cells;
abscissa=different final concentrations of iota-, kappa- or
lambda-carrageenan in the virus suspension in μg/ml.

[0102] Ordinate=percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-carrageenan (dark bars) and fucoidan (light bars) in the virus
suspension in μg/ml.

[0104] Ordinate=percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-, kappa- or lambda-carrageenan or fucoidan in the virus suspension
in μg/ml.

[0106] Ordinate-percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-, kappa- or lambda-carrageenan or fucoidan in the virus suspension
in μg/ml.

[0107] FIG. 15 shows the efficacy of iota-, kappa-, lambda-carrageenan and
fucoidan in reducing plaque formation of influenza A H5N1 virus in MDCK
cells at doses of 400 μg/ml and 44 μg/ml.

[0108] Ordinate=percentage of plaque formation after infection of MDCK
cells with avian influenza H5N1 virus suspension containing different
concentrations of iota-, kappa-, lambda-carrageenan or fucoidan relative
to the plaque formation of MDCK cells infected with influenza H5N1 virus
suspension without polymer (set as 100%); abscissa=different final
concentrations of iota-, kappa-, lambda-carrageenan or fucoidan in the
virus suspension in μg/ml.

[0112] Ordinate-percentage of uninfected cells after infection relative to
uninfected control cells; abscissa=different final concentrations of
iota-carrageenan in the virus suspension (dark bars) or in the
preincubation medium (light bars) in μg/ml; dark bars=cells infected
with Ad50 in the presence of iota-carrageenan; light bars=cells
preincubated for 3 h with iota-carrageenan, subsequently washed three
times with PBS and infected with Ad50 in the absence of iota-carrageenan.

[0113] In order that the invention described herein may be more fully
understood, the following examples are set forth. The examples are for
illustrative purposes only and are not to be construed as limiting this
invention in any respect. It is further understood that the present
invention shall also comprise variations of the expressly disclosed
embodiments to an extent as would be contemplated by a person of ordinary
skill in the art.

EXAMPLES

Example 1

Effect of Different Concentrations of Iota-Carrageenan on Influenza A
Virus Plaque Formation in MDCK Cells

[0114] Virus suspensions containing 60-80 pfu of influenza virus
A/Chile/1/93 H1N1 were mixed with a iota-carrageenan stock solution to
final concentrations of 0.1, 1, 10, 25, 50 or 100 μg/ml. Confluent
monolayers of the canine kidney cell line MDCK in six well plates were
infected with the virus suspensions for 60 min at 34° C. The
infection inoculum was removed and cells were washed with PBS and agarose
overlay containing 0.6% agarose was added. Plates were incubated at
36° C. in a humidified atmosphere of 5% CO2 in air. 48-60 h
after infection the agarose overlay was removed, cells were stained with
crystal violet stain and visible plaques were counted. The percentage of
plaque formation relative to the infected control (without
iota-carrageenan treatment) was determined for each iota-carrageenan
concentration.

[0115] As shown in FIG. 1, it was found that iota-carrageenan inhibits, in
a dose dependent manner, the plaque formation of influenza A/Chile/1/93
H1N1 virus in MDCK cells. A 50% reduction in plaque number (IC50) was
achieved at a iota-carrageenan concentration of 50 μg/ml.

Example 2

Effect of Different Concentrations of Iota-Carrageenan on Influenza A
Virus Plaque Formation in MDCK Cells

[0116] Virus suspensions containing 60-80 pfu of influenza virus
A/Aichi2/68 H3N2 were mixed with a iota-carrageenan stock solution to
final concentrations of 75, 150 or 300 μg/ml. Confluent monolayers of
the canine kidney cell line MDCK in six well plates were infected with
the virus suspensions for 60 min at 34° C. The infection inoculum
was removed and cells were washed with PBS and agarose overlay containing
0.6% agarose was added. Plates were incubated at 37° C. in a
humidified atmosphere of 5% CO2 in air. 48-60 h after infection the
agarose overlay was removed, cells were stained with crystal violet stain
and visible plaques were counted. The percentage of plaque formation
relative to the infected control (without iota-carrageenan) was
determined for each iota-carrageenan concentration.

[0117] As shown in FIG. 2, it was found that iota-carrageenan inhibits, in
a dose dependent manner, the plaque formation of influenza A/Aichi/2/68
H3N2 virus in MDCK cells.

Example 3

Effect of Different Concentrations of Iota-Carrageenan on Parainfluenza
Virus 3 Plaque Formation in Hep-2 Cells

[0118] Virus suspensions containing 60-80 pfu of parainfluenza virus 3
were mixed with a iota-carrageenan stock solution to final concentrations
of 0.1, 1, 10, 25, 50 and 100 μg/ml. The mixture was incubated for 1 h
at 34° C.

[0119] Confluent monolayers of Hep-2 cells in six well plates were
infected with the virus suspensions for 60 minutes a 34° C. The
infection inoculum was removed and cells were washed with PBS and agarose
overlay containing 0.6% agarose was added. The trays were incubated in a
humidified, 5% CO2 atmosphere. 48-60 h after infection the agraose
overlay was removed, cells were stained with crystalviolett stain and
visible plaques were counted. The percentage of plaque formation relative
to the infected control (without iota-carrageenan) plates was determined
for each iota-carrageenan concentration.

[0120] As shown in FIG. 3, it was found that iota-carrageenan inhibits, in
a dose dependent manner, the plaque formation of parainfluenza virus 3 in
Hep-2 cells. A 50% reduction in plaque number (IC50) was achieved at an
iota-carrageenan concentration of 10 μg/ml.

[0121] Confluent monolayers of HeLa cells were incubated three hours with
iota-carrageenan at a concentration of 13, 40, 133 and 400 μg/ml. The
iota-carrageenan containing supernatant was removed and the cells were
washed three times with PBS and thereafter infected with parainfluenza
virus 3 as described in Example 3, but without addition of
iota-carrageenan to the virus suspension. The percentage of plaque
formation relative to the infected control (without iota-carrageenan
pretreatment) was determined for each iota-carrageenan concentration.

[0122] As shown in FIG. 4, it was found that iota-carrageenan inhibits the
plaque formation of parainfluenza virus 3 at a concentration of 400 and
133 μg/ml when cells were preincubated for three hours with
iota-carrageenan, although no iota-carrageenan was present at the time of
the infection and during the entire subsequent incubation period at
37° C. This result indicates that iota-carrageenan chemically or
structurally modifies the surface receptors of the host cell in a way
such that receptor-mediated binding of parainfluenza virus 3 to the host
cell is hindered or prevented even in the absence of the modifying agent
carrageenan. This is also proof of a strong prophylactic efficacy of
iota-carrageenan.

Example 5

Effect of Eukaryotic Cell Pretreatment by Different Carrageenans on the
Inhibition of Coronavirus Mediated Cell Death

[0123] Subconfluent CK cells were infected in the presence of iota-,
kappa- or lambda carrageenan at a concentration of 4, 40 and 400 μg/ml
with feline coronavirus FIPV at a moi (multiplicity of infection) of 0.1)
(see FIG. 5A). In comparison, subconfluent CK cells were incubated three
hours with iota-, lambda or kappa-carrageenan at a concentration of 4,
40, and 400 μg/ml. The carrageenan containing supernatant was removed
and the cells were washed three times with PBS and infected with feline
coronavirus FIP (moi=0.1) in the absence of the polymer (see FIG. 5B).
The percentage of viable cells relative to the uninfected control was
determined for each carrageenan and each concentration.

[0124] As shown in FIG. 5A, all three types of carrageenan inhibit the
coronavirus mediated cell death in CK cells at the highest concentration
of 400 μg/ml. Iota-carrageenan still shows significant inhibition at a
concentration of 4 μg/ml while kappa- and lambda-carrageenan are not
effective at this concentration. From FIG. 5B it can be taken that in
contrast to viruses that enter the cell via sugar receptors (see
preceding examples), coronavirus infection does not seem to be inhibited
due to a chemical or structural modification of the coronavirus-specific
receptor(s) at the host cell surface by carrageenan, since it was not
possible to increase host cell protection beyond a level of 35%
inhibition even at the highest experimental carrageenan concentration of
400 μg/ml and at a preincubation period of three hours. Nor did the
pretreatment of the host cells with carrageenan significantly improve
cell protection against coronavirus infection. The results suggest that
in order to achieve a significant protection against coronavirus
infection the antiviral active agent, i.e. carrageenan, must be present
at the time of infection, i.e. when an interaction between the virus and
the host cell is about to occur.

Example 6

Effect of Eukaryotic Cell Pretreatment by Iota-Carrageenan on the
Inhibition of HRV8 Mediated Cell Death

[0125] Subconfluent HeLa cells were infected with human rhinovirus type 8
(HRV8, moi=0.1) in the presence of iota-carrageenan at concentrations of
4, 40 and 400 μg/ml (see FIG. 6A). In comparison, subconfluent HeLa
cells were incubated for three hours with iota-carrageenan at a
concentration of 4, 40 and 400 μg/ml prior to infection. The
supernatant containing iota-carrageenan was removed and the cells were
washed three times with PBS and then infected with HRV8 in the absence of
the polymer (see FIG. 6B). The percentage of viable cells relative to the
uninfected control was determined for each iota-carrageenan
concentration.

[0126] As shown in FIG. 6A, iota-carrageenan inhibits the HRV8 mediated
cell death at all concentrations. From FIG. 6B it can be taken that, in
contrast to viruses that enter the cell via sugar receptors, HRV8
mediated cell death was not inhibited by more than 5% even at 400
μg/ml and where cells were preincubated for three hours with
iota-carrageenan. This result is consistent with previous findings (data
not shown) and suggests that for HRV8 the preincubation of the target
cells with carrageenan does not significantly protect the target cells
from becoming infected if carrageenan is missing at the time of
infection. This result indicates that the receptor for HRV8, the
LDL-receptor, is not modified or masked by the treatment with
iota-carrageenan.

Example 7

Iota-Carrageenan is Quantitatively Removed from HeLa Cells by Washing the
Cells with PBS

[0127] Hela cells were incubated with FITC-labelled iota-carrageenan at a
concentration of 400, 133, 4 and 0.4 μg/ml for 10 minutes, then the
supernatant was removed and the cells were washed three times with PBS.
The amount of residual FITC-labelled carrageenan was determined with a
fluorescent detection reader (BMG-Omega) after removing the supernatant
containing FITC-labelled iota-carrageenan and after each washing step.
The fluorescent light units were converted into concentration values of
μg/ml carrageenan by using a standard curve.

[0128] As shown in FIG. 7 FITC-labelled iota-carrageenan was
quantitatively removed (>95%) from HeLa cells by washing the cells at
least three times with PBS. This result proves that carrageenan does not
covalently bind to the surface of the cells. The result was confirmed by
a second experimental set, where the incubation period was extended from
10 minutes to 3 hours using the same detection method. The results thus
support the conclusions drawn herein and in the examples concerned that
carrageenan induces a modification of the sugar receptors on the target
cell surface involved in the binding of the virions, which modification
is still present after removal of the polymer from the target cell
surface.

Example 8

Effect of Eukaryotic Cell Pretreatment by Iota-Carrageenan on the
Inhibition of RSV Mediated Cell Death

[0129] Subconfluent Vero cells were infected in the presence of
iota-carrageenan at concentrations of 0.4, 4 and 40 μg/ml with RSV
(moi=0.1). In comparison, subconfluent Vero cells were incubated for
three hours with iota-carrageenan at a concentration of 0.4, 4 and 40
μg/ml. The iota-carrageenan containing supernatant was removed and the
cells were washed three times with PBS and infected with RSV (moi=0.1) in
the absence of the carrageenan. The percentage viable cells relative to
the uninfected control was determined for each carrageenan concentration.

[0130] As shown in FIG. 8, iota-carrageenan inhibits the RSV mediated cell
death at all concentrations even if the polymer is absent at the time of
and during infection. It is known that RSV attaches to the cells via
heparan sulphate, a sugar molecule that is present on the surface of
epithelial cells. The results indicate that this receptor is modified by
iota-carrageenan and the attachment of RSV to the cell surface and the
subsequent viral replication is thereby blocked.

Example 9

Effect of Eukaryotic Cell Treatment with Iota-Carrageenan at Different
Time Points After Infection on Inhibition of RSV Mediated Cell Death in
HNep Cells

[0131] Subconfluent HNep cells were infected with RSV (moi=0.1).
Iota-carrageenan was added at a final concentration of 40 μg/ml at
different time points post infection as indicated in FIG. 9 (0, 8, 24,
32, 48, 56 and 72 h post infection) and the percentage of viable HNep
cells relative to the uninfected control was determined for each time
point.

[0132] As shown in FIG. 9, iota-carrageenan significantly inhibits the RSV
mediated cell death at a concentration of 100 μg/ml even when the
polymer was added only 24 hours after infection. This result demonstrates
that iota-carrageenan cannot only be used prophylactically but may also
exert antiviral efficacy in the course of a therapeutic treatment when
applied in the early phase of viral infection.

Example 10

Effect of Prophylactic Treatment with Iota-Carrageenan on Inhibition of
RSV Mediated Cell Death in HNep Cells

[0133] Subconfluent HNep cells were infected in the presence of
iota-carrageenan at a concentration of 400, 133, 44, 15, 5, 2 and 1
μg/ml with RSV (moi=0.1). The percentage of viable HNep cells relative
to an uninfected control was determined for each iota-carrageenan
concentration.

[0134] As shown in FIG. 10, iota-carrageenan significantly inhibits the
RSV mediated cell death even at a concentration as low as 1 μg/ml when
the polymer was present during infection. This result demonstrates that
iota-carrageenan can be used effectively for prophylactic intervention
strategies.

Example 11

Effect of Eukaryotic Cell Treatment by Different Carrageenans on the
Inhibition of RSV Mediated Cell Death in HEp-2 Cells

[0135] HEp-2 cells in 6 well plates were infected in the presence of
iota-, kappa- or lambda carrageenan at a concentration of 0.01, 0.1, 1,
10 and 100 μg/ml with RSV A2 virus (moi=0.001). The percentage of
viable cells relative to the uninfected control was determined for each
carrageenan and each concentration.

[0136] As shown in FIG. 11, all three types of carrageenan inhibit the RSV
mediated cell death in HEp-2 cells with iota-carrageenan showing the
strongest effect.

Example 12

Comparison of the Effect of Eukaryotic Cell Treatment by Iota-Carrageenan
and Fucoidan on the Inhibition of RSV Mediated Cell Death

[0137] Subconfluent HNep cells were infected in the presence of
iota-carrageenan and fucoidan at a concentration of 400, 133, 44, 15, 4,
1.3 and 0.4 with RSV (moi=0.1). The percentage of viable HNep cells
relative to an uninfected control was determined for each
iota-carrageenan and each fucoidan concentration.

[0138] As shown in FIG. 12, fucoidan significantly inhibits the RSV
mediated cell death in HNep cells at a concentration of 0.4 μg/ml when
the polymer is present during infection. Fucoidan is therefore an
interesting candidate for the development of products for prophylaxis and
treatment of RSV infections.

Example 13

Effect of Eukaryotic Cell Treatment with Different Carrageenans and
Fucoidan on the Inhibition of Parainfluenza Virus 3 Mediated Cell Death

[0139] Subconfluent HNep cells were infected in the presence of either
iota-, kappa-, lambda-carrageenan or fucoidan at a concentration of 400,
133.3, 44.4, 14.8, 4, 1.3 and 0.4 μg/ml with parainfluenza virus type
3. The percentage of viable (hence uninfected) HNep cells relative to the
uninfected control was determined for each polymer and each polymer
concentration.

[0140] As shown in FIG. 13, all three types of carrageenan inhibit the
parainfluenza virus 3 mediated cell death in HNep cells with
iota-carrageenan showing the strongest effect. Fucoidan also inhibits the
parainfluenza virus 3 mediated cell death in HNep cells at an extent
comparable to the inhibitory effect of kappa- and lambda-carrageenan.

Example 14

Effect of Eikaryotic Cell Treatment with Different Carrageenans and
Fucoidan on the Inhibition of Influenza Virus H3N2 Mediated Cell Death

[0141] Subconfluent HNep cells were infected in the presence of iota-,
kappa-, lambda-carrageenan and fucoidan at a concentration of 400, 133.3,
44.4, 14.8, 4, 1.3 and 0.4 μg/ml with influenza virus H3N2. The
percentage of viable HNep cells relative to the uninfected control was
determined for each polymer and each polymer concentration.

[0142] As shown in FIG. 14, all three types of carrageenan inhibit the
influenza virus H3N2 mediated cell death in HNep cells. In addition, it
was found that fucoidan inhibits the influenza virus H3N2 mediated cell
death in HNep at an extent comparable to that of iota-carrageenan.

Example 15

Effect of Different Carrageenans and Fucoidan on Avian Influenza Virus
H5N1 Plaque Formation in MDCK Cells

[0143] Virus suspensions containing 60-80 pfu of avian influenza virus
H5N1 were mixed with a polymer stock solution of either iota-, kappa-,
lambda-carrageenan or fucoidan to final polymer concentrations of 400 or
44.4 μg/ml. Confluent monolayers of the canine kidney cell line MDCK
in six well plates were infected with the virus suspensions for 60 min at
34° C. The infection inoculum was removed and cells were washed
with PBS and agarose overlay containing 0.6% agarose was added. Plates
were incubated at 36° C. in a humidified atmosphere of 5% CO2
in air. 48-60 h after infection the agarose overlay was removed, cells
were stained with crystal violet stain and visible plaques were counted.
The percentage of plaque formation relative to the infected control
(without polymer treatment) was determined for each polymer and each
polymer concentration.

[0144] As shown in FIG. 15, it was found that plaque formation of avian
influenza virus H5N1 was not influenced by iota- and lambda carrageenan
and fucoidan. However, kappa-carrageenan inhibits, in a dose dependent
manner, the plaque formation of avian influenza virus H5N1 in MDCK cells.
Since avian influenza virus preferably binds to sialic acid residues with
alpha 2-3 linkage the results indicate that kappa-carrageenan may
preferably modify such sialic acid residues having 2-3 linkage.

Example 16

Effect of Prophylactic Eukaryotic Cell Treatment with Iota-Carrageenan on
the Inhibition of Adenovirus Type B (Ad50) Mediated Cell Death

[0145] Subconfluent HNep cells were infected in the presence of
iota-carrageenan at a concentration of 400, 133.3, 44.4, 14.8, 4, 1.3 and
0.4 μg/ml with Ad50. The percentage of viable HNep cells relative to
the uninfected control was determined for each iota-carrageenan
concentration.

[0146] As shown in FIG. 16, iota-carrageenan significantly inhibits the
Ad50 mediated cell death even at a concentration as low as 4 μg/ml
when the polymer is present at the time of and during infection. This
result is an indication that iota-carrageenan may be used for effective
prophylactic intervention strategies against adenovirus of the subtype B
(e.g. Ad50). However, when other adenoviruses from subtypes A, C and D
were tested in an experimental set as described above no significant
effect of iota-carrageenan was detected (data not shown).

Example 17

Comparison of the Effect of Prophylactic Eukaryotic Cell Treatment with
Iota-Carrageenan on the Inhibition of Ad50 Mediated Cell Death

[0147] Subconfluent HNep cells were infected in the presence of
iota-carrageenan at a concentration of 400, 40 and 4 μg/ml with Ad50.
In comparison, subconfluent HNep cells were incubated for three hours
with iota-carrageenan at a concentration of 400, 40 and 4 μg/ml prior
to infection. The supernatant containing iota-carrageenan was removed and
the cells were washed three times with PBS and infected with Ad50 in the
absence of the carrageenan. The percentage of viable HNep cells relative
to the uninfected control was determined for each iota-carrageenan
concentration.

[0148] As shown in FIG. 17, iota-carrageenan significantly inhibits the
Ad50 mediated cell death at a concentration of 400 and 40 μg/ml in the
presence and in the absence of iota-carrageenan at the time of infection
and during the observed infection period. These data indicate that
iota-carrageenan modifies the cell surface receptor for adenoviruses from
subtype B, which is known to be a sugar receptor, while adenoviruses from
other subtypes probably enter the cell via different receptors.

Patent applications by Andreas Grassauer, Vienna AT

Patent applications by Eva Prieschl-Grassauer, Vienna AT

Patent applications by MARINOMED BIOTECHNOLOGIE GMBH

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